Characterization of the simultaneous decay kinetics of metarhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy

Jochen Stehle, Robert Silvers, Karla Werner, Deep Chatterjee, Santosh Gande, Frank Scholz, Arpana Dutta, Josef Wachtveitl, Judith Klein-Seetharaman, Harald Schwalbe

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

The mammalian visual dim-light photoreceptor rhodopsin is considered a prototype Gprotein-coupled receptor. Here, we characterize the kinetics of its light-activation process. Milligram quantities of α,ε- 15N-labeled tryptophan rhodopsin were produced in stably transfected HEK293 cells. Assignment of the chemical shifts of the indole signals was achieved by generating the single-point-tryptophan to phenylalanine mutants, and the kinetics of each of the five tryptophan residues were recorded. We find kinetic partitioning in rhodopsin decay, including three half-lives, that reveal two parallel processes subsequent to rhodopsin activation that are related to the photocycle. The metaII and metaIII states emerge in parallel with a relative ratio of about 3:1. Transient formation of the metaIII state was confirmed by flash photolysis experiments. From analysis of the site-resolved kinetic data we propose the involvement of the E2-loop in the formation of the metaIII state.

Original languageEnglish (US)
Pages (from-to)2078-2084
Number of pages7
JournalAngewandte Chemie - International Edition
Volume53
Issue number8
DOIs
StatePublished - Feb 17 2014
Externally publishedYes

Keywords

  • bovine rhodopsin
  • G-protein coupled receptors
  • membrane protein dynamics
  • NMR spectroscopy
  • photocycles

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)

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